ABSTRACT
Chemokines induce calcium (Ca(2+)) signaling and chemotaxis in dendritic cells (DCs), but the molecular players involved in shaping intracellular Ca(2+) changes remain to be characterized. Using siRNA and knockout mice, we show that in addition to inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release and store-operated Ca(2+) entry (SOCE), the transient receptor potential melastatin 2 (TRPM2) channel contributes to Ca(2+) release but not Ca(2+) influx in mouse DCs. Consistent with these findings, TRPM2 expression in DCs is restricted to endolysosomal vesicles, whereas in neutrophils, the channel localizes to the plasma membrane. TRPM2-deficient DCs show impaired maturation and severely compromised chemokine-activated directional migration as well as bacterial-induced DC trafficking to the draining lymph nodes. Defective DC chemotaxis is due to perturbed chemokine-receptor-initiated Ca(2+) signaling mechanisms, which include suppression of TRPM2-mediated Ca(2+) release and secondary modification of SOCE. DCs deficient in both TRPM2 and IP(3) receptor signaling lose their ability to perform chemotaxis entirely. These results highlight TRPM2 as a key player regulating DC chemotaxis through its function as Ca(2+) release channel and confirm ADP-ribose as a novel second messenger for intracellular Ca(2+) mobilization.
Subject(s)
Calcium/metabolism , Chemotaxis/physiology , Dendritic Cells/cytology , Dendritic Cells/physiology , Lysosomes/metabolism , TRPM Cation Channels/metabolism , Adenosine Diphosphate Ribose , Animals , Calcium Signaling/physiology , Chemokines/pharmacology , Dendritic Cells/drug effects , Gene Expression Regulation/physiology , Inflammasomes/metabolism , Inositol 1,4,5-Trisphosphate Receptors/genetics , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Mice , Mice, Inbred C57BL , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small InterferingABSTRACT
The ectoenzyme CD38 catalyzes the production of cyclic ADP-ribose (cADPR) and ADP-ribose (ADPR) from its substrate, NAD(+). Both products of the CD38 enzyme reaction play important roles in signal transduction, as cADPR regulates calcium release from intracellular stores and ADPR controls cation entry through the plasma membrane channel TRPM2. We previously demonstrated that CD38 and the cADPR generated by CD38 regulate calcium signaling in leukocytes stimulated with some, but not all, chemokines and controls leukocyte migration to inflammatory sites. However, it is not known whether the other CD38 product, ADPR, also regulates leukocyte trafficking In this study we characterize 8-bromo (8Br)-ADPR, a novel compound that specifically inhibits ADPR-activated cation influx without affecting other key calcium release and entry pathways. Using 8Br-ADPR, we demonstrate that ADPR controls calcium influx and chemotaxis in mouse neutrophils and dendritic cells activated through chemokine receptors that rely on CD38 and cADPR for activity, including mouse FPR1, CXCR4, and CCR7. Furthermore, we show that the calcium and chemotactic responses of leukocytes are not dependent on poly-ADP-ribose polymerase 1 (PARP-1), another potential source of ADPR in some leukocytes. Finally, we demonstrate that NAD(+) analogues specifically block calcium influx and migration of chemokine-stimulated neutrophils without affecting PARP-1-dependent calcium responses. Collectively, these data identify ADPR as a new and important second messenger of mouse neutrophil and dendritic cell migration, suggest that CD38, rather than PARP-1, may be an important source of ADPR in these cells, and indicate that inhibitors of ADPR-gated calcium entry, such as 8Br-ADPR, have the potential to be used as anti-inflammatory agents.
Subject(s)
ADP-ribosyl Cyclase 1/metabolism , Adenosine Diphosphate Ribose/analogs & derivatives , Adenosine Diphosphate Ribose/pharmacology , Bone Marrow Cells/drug effects , Chemotaxis/drug effects , Neutrophils/drug effects , ADP-ribosyl Cyclase 1/deficiency , Adenosine Diphosphate Ribose/chemical synthesis , Adenosine Diphosphate Ribose/chemistry , Animals , Bone Marrow Cells/immunology , Calcium/antagonists & inhibitors , Calcium/immunology , Cell Line , Chemotaxis/immunology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Leukocytes/drug effects , Leukocytes/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , NAD/analogs & derivatives , NAD/pharmacology , Neutrophils/immunology , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/immunology , Sensitivity and Specificity , Structure-Activity Relationship , Time FactorsABSTRACT
Melastatin-related TRPM ion channels have emerged as novel therapeutic targets due to their potential ability to modulate the function and fate of immune cells during inflammation, innate, and adaptive immunity. Four family members, TRPM1, TRPM2, TRPM4 and TRPM7 have a strong presence in the immune system. TRPM channels regulate ion-homeostasis by sensing cellular redox status and cytoplasmic calcium levels. TRPM2 for example, is highly expressed in phagocytes. This channel is activated by intracellular ADP-ribose upon exposure to oxidative stress and induces cell death. Here we will review the functional links between TRPM-mediated ion conductance, chemotaxis, apoptosis, and innate immunity.
